Sbp1p affects translational repression and decapping in Saccharomyces cerevisiae

Abstract

The relationship between translation and mRNA turnover is critical to the regulation of gene expression. One major pathway for mRNA turnover occurs by deadenylation, which leads to decapping and subsequent 5'-to-3' degradation of the body of the mRNA. Prior to mRNA decapping, a transcript exits translation and enters P bodies to become a potential decapping substrate. To understand the transition from translation to decapping, it is important to identify the factors involved in this process. In this work, we identify Sbp1p (formerly known as Ssb1p), an abundant RNA binding protein, as a high-copy-number suppressor of a conditional allele in the decapping enzyme. Sbp1p overexpression restores normal decay rates in decapping-defective strains and increases P-body size and number. In addition, Sbp1p promotes translational repression of mRNA during glucose deprivation. Moreover, P-body formation is reduced in strains lacking Sbp1p. Sbp1p acts in conjunction with Dhh1p, as it is required for translational repression and P-body formation in pat1Delta strains under these conditions. These results identify Sbp1p as a new protein that functions in the transition of mRNAs from translation to an mRNP complex destined for decapping.

FIG. 1.

Sbp1p overexpression suppresses the full-length (F.L.) to pG fragment (Frag.) defect. The ratio of full-length MFA2pG reporter to the pG fragment is shown and is a simple measurement of decapping activity. (a) Steady-state distributions in strains containing dcp1-2 at the nonpermissive temperature of 33°C. (b) Steady-state distributions in strains containing dcp2-7 at the nonpermissive temperature of 37°C. (c) mRNA distributions for strains with mutations in the decapping enzyme (dcp1Δ and dcp2Δ) or strains with mutations in the decapping activators (dhh1Δ, lsm1Δ, and pat1Δ). (d) Steady-state distributions for dhh1Δ pat1Δ and dhh1Δ pat1Δ SBP1 2μm strains. Zero denotes less than 5% fragment. The temperatures at which the experiments were performed are noted next to the panels.

FIG. 2.

Sbp1p overexpression suppresses mRNA half-life (t_1/2) defects in conditional decapping mutants. Agarose Northern blot assays were done for the MFA2pG reporter mRNAs for the wild-type (a), SBP1 2μm (b), dcp1-2 (c), and dcp1-2 SBP1 2μm (d) strains at 33°C and for the wild-type (e), SBP1 2μm (f), dcp2-7 (g), and dcp2-7 SBP1 2μm (h) strains at 37°C. Time points after transcriptional repression are indicated above the lanes. The top band in each panel is full-length MFA2pG mRNA, and the bottom band is the pG fragment. The 7S RNA shown below each panel was used as a loading control. Half-lives were determined with the full-length MFA2pG mRNA band and are indicated beside the panels. All experiments were done a minimum of three times.

FIG. 3.

Sbp1p is not required for normal rates of mRNA decay. Both MFA2pG and PGK1pG mRNA decay rates in both wild-type and sbp1Δ mutant strains are shown. Time points after transcriptional repression are shown above the lanes. The 7S RNA shown was used as a loading control. Half-lives (t_1/2) are indicated beside the panels. All experiments were done a minimum of three times.

FIG. 4.

Sbp1p localizes to P bodies under stress conditions. SBP1-GFP cells were grown to log phase and resuspended in minimal (min) medium containing dextrose (a), grown to log phase and resuspended in minimal (min) medium lacking dextrose (b), or grown to a high OD (OD, ∼12) (c). (d to f) SBP1-GFP cells containing the DCP2-RFP plasmid were grown to a high OD (OD ∼12). Panels: d, Sbp1p-GFP; e, Dcp2p-RFP; f, merge.

FIG. 5.

sbp1Δ fails to repress translation after glucose deprivation when put in combination with pat1Δ but not dhh1Δ. Shown are polysome profiles (OD, A₂₆₀) of the yeast strains grown continuously in glucose (+glucose) and after glucose deprivation (−glucose). Panels: a, wild type; b, sbp1Δ; c, pat1Δ; d, pat1Δ sbp1Δ; e, dhh1Δ; f, dhh1Δ sbp1Δ. All experiments were done a minimum of three times.

FIG. 6.

Translation rate is affected in sbp1Δ strains after glucose deprivation. Incorporation of [³⁵S]methionine in various yeast strains grown continuously in glucose (diamonds) and after glucose deprivation (squares) in wild-type (a), sbp1Δ (b), pat1Δ (c), pat1Δ sbp1Δ (d), dhh1Δ (e), and dhh1Δ sbp1Δ (f) strains. Time is marked on the x axis in minutes, and incorporation is marked on the y axis in counts per minute. The percent decrease in the [³⁵S]methionine incorporation rate of cells resuspended in medium lacking glucose compared to that of cells grown continuously in glucose is noted next to each genotype. All experiments were done a minimum of three times.

FIG. 7.

Sbp1p and Pat1p have additive effects on P-body formation. With Dcp2p-GFP as a marker for P bodies, we observed P-body formation at a high OD in wild-type (a), sbp1Δ (b), pat1Δ (c), pat1Δ sbp1Δ (d), dhh1Δ (e), and dhh1Δ sbp1Δ (f) strains.

FIG. 8.

Overexpression of Sbp1p causes translational repression. Shown are polysome profiles from control cells carrying an empty vector (pGAL-empty) and cells carrying an Sbp1p overexpression vector (pGAL-SBP1). pGAL-empty cells grown in sucrose (suc) (a), pGAL-empty cells grown in galactose (gal) (b), pGAL-SBP1 cells grown in sucrose (c), and pGAL-SBP1 cells grown in galactose to induce Sbp1p overexpression (d) were tested. All experiments were done a minimum of three times.

FIG. 9.

Sbp1p overexpression affects the number and size of P bodies. SBP1 was overexpressed in strains carrying endogenous GFP-tagged markers for P bodies. Panels: a, DHH1-GFP; b, DHH1-GFP SBP1 2μm; c, DCP2-GFP; d, DCP2-GFP 2μm.